Gelation is an important functional property of proteins as it provides texture and support in foods. Generally, thermal gelation of globular proteins involves unfolding of the protein molecules by heating, which leads to exposure of hydrophobic amino acid residues. Later, unfolded molecules re-arrange and aggregate irreversibly via disulfide bridges, hydrogen bonds, hydrophobic and/or van der Waals interactions. Finally, aggregation carries on with association of protein particles and if the protein concentration is sufficiently high, a three-dimensional network is created (Lefèvre & Subirade, 2000). This process only takes place in the presence of suitable environmental conditions, such as pH, temperature and ionic strength (Totosaus, Montejano, Salazar, & Guerrero, 2002). (Twomey, Keogh, Mehra, & O'Kennedy, 1997, Ziegler, & Foegeding, 1990).
Plant proteins are normally considered inferior to animal proteins in terms of gelling properties. Gelatin, egg white and whey proteins are widely used as gelling agents in the food industry, particularly in meat and dairy based systems. In recent years, proteins derived from plant sources are becoming an important ingredient segments owing to health (no Bovine Spongiforme Encephalopathy concern), religious and cost reasons. For a long time, soy protein has been the major plant protein gelling ingredient in the market. Yet there is an opportunity for other novel gelling ingredients of plant origin to meet the increasing market requirement for different functionalities and sensory attributes.
Oat is commonly used as an animal feed and only a small percentage of the grain is currently used for human consumption. Recently the human food market for oat has been gaining momentum mainly due to the growing public awareness of the health benefits of β-glucan. This soluble dietary fiber component of oat is known to reduce blood cholesterol (Braaten, Wood, & Scott, 1994), and regulate blood glucose levels (Wood, Scott, Riedel, Wolynetz, & Collins, 1994). Several techniques have been developed to isolate β-glucan from oat grain as a health ingredient in food products. The remaining components such as protein and starch are awaiting research to develop their full value (Inglett, Lee, & Stevenson, 2008).
Oat has the highest protein level (12-20%) (Mohamed, Biresaw, Xu, Hojilla-Evangelista, & Rayas-Duarte, 2009) among cereals, with a superior amino acid profile due to higher amounts of limiting amino acids lysine and threonine (Klose & Arendt, 2012). This is related to the fact that in most cereals the major storage proteins are alcohol-soluble prolamines whereas in oat, globulins represent 70-80% of the total protein fraction (Robert, Nozzolillo, Cudjoe, & Altosaar, 1983). The major fraction in oat protein is the 12S globulin, which consists of two major subunits with molecular weight of about 32 and 22 kDa called the A- and B-subunits, where the A-subunit is an acidic polypeptide and the B-subunit is a basic polypeptide. The A- and B-subunits are disulfide bonded in the native globulin, forming a dimer with a molecular weight of 54 kDa, which further associates into a hexamer through noncovalent forces (Burgess, Shewry, Matlashewski, Altosaar, & Miflin, 1983). The 7S and 3S are the minor fractions. 7S globulins are polypeptides with molecular weight of 55 kDa, and some minor components with a molecular weight of 65 kDa are also present. The 3S fraction entails at least two major components with molecular weight of about 15 and 21 kDa (Klose & Arendt, 2012).
Two previous publications demonstrated that oat protein could form gels (Ma & Harwalkar, 1987; Ma, Khanzada, & Harwalkar, 1988). But at acidic and neutral pH levels, very weak gels with poor water holding capacity were obtained. The gel properties improved above about pH 8, but strong gels could only be prepared at pHs 9-10. The gel hardness was greatly increased by both acetylation and succinylation (Ma & Wood, 1986, 1987). The authors suggested that the changes in the functional properties of oat protein after modification resulted from altered conformation and increase in net charge (Ma, 1984, 1985; Ma & Wood, 1986, 1987). This was later confirmed with the study of the thermal aggregation of oat globulin by Raman spectroscopy (Ma, Rout, & Phillips, 2003). In this work, changes in protein interactions and conformation were induced by the addition of protein structure modifying agents such as chaotropic salts, sodiumdodecyl sulfate or dithiothreitol, which can either enhance or inhibit thermal gelation of oat globulin.
Enzymatic hydrolysis is a preferable tool to alter functional properties of proteins because of milder processing conditions required, easier control of reaction and minimal formation of by-products (Mannheim & Cheryan, 1992). Recent research has reported the effect of enzymatic hydrolysis over the gelling properties of proteins including soy protein (Hou & Zhao, 2011), rice bran protein (Yeom, Lee, Ha, Ha, & Bae, 2010), sunflower protein (Sanchez & Burgos, 1997), and canola protein (Pinterits & Arntfield, 2007). Results from these studies indicate that improvement of the gelling capacity is highly enzyme specific. The gelling properties of oat protein treated with trypsin were studied in previous work (Ma & Wood, 1986, 1987), however, weak gel structure was obtained due to the short size of the protein molecules, which may no longer be able to associate to form a strong gel matrix. Since the final composition and thus the use of the hydrolysates will depend on the type of enzyme used and the hydrolysis conditions (Benítez, Ibarz, & Pagan, 2008), a systematic investigation of the effect of various proteases over the gelling capacity of oat protein is required. Such information has not been available, however important for the development of new modification strategy to improve oat protein gelling properties.
Modification of protein conformation can also be achieved through limited hydrolysis, as changes in the secondary and tertiary structure can be produced. This can alter the surface exposure of reactive amino acids, leading to an increase in interactions favoring aggregation (Foegeding & Davis, 2011) and three-dimensional network formation.
Cold-set gelation as alternative gelling method opens an interesting opportunity for proteins in development of functional food ingredient, such as protecting heat sensitive bioactive compounds. This process consists of two consecutive steps. The first step is preheating protein above denaturation temperature to induce protein unfolding, exposure of reactive groups, and subsequent aggregation at solution pH far from protein isoelectric point (IEP) and at a concentration below a critical value. In this step, protein remains as soluble aggregates due to the high electrostatic repulsive forces. For the second step, addition of salt (Ca2+) or altering solution pH induce the formation of three-dimensional gel network (Bryant and McClements, 1998, Alting, de Jongh, Visschers, & Simons, 2002; Alting, Hammer, de Kruif, & Visschers, 2003a; Campbell, Gu, Dewar, & Euston, 2009). Generally, two kinds of cold-set gels, particulate and filamentous gels, can be achieved depending on processing conditions (Lefèvre, and Subirade, 2000; Maltais, Remondetto, Gonzalez, Subirade, 2005; Maltais, Remondetto, Subirade, 2008): Filamentous gel is formed by linearly linked protein aggregates maintained by hydrophobic interactions at low ionic strength or pH far from protein IEP, which exhibits regular network structure with more or less linear strands. In contrast, particulate gel is created by random aggregation of protein units mainly through van der Waals interaction at high ionic strength or pH near protein IEP, which composes of large and almost spherical aggregates. These different predominated interactions and gel network structures lead to various gel mechanical properties and applications (Remondetto, Neyssac, & Subirade, 2004).
Extensive works have focused on salt-induced whey protein and soy protein gels in terms of gel properties, formation mechanism and applications (Maltais, Remondetto, Subirade. 2010; Barbut, & Foegeding, 1993, Foff, and Roegeding, 1996; Zhang, Liang, Chen, Subirade, 2012). These cold-set gels were used to improve the texture and stability of food products (Hongsprabhas, & Barbut, 1999), or play as carrier of bioactive compounds or divalent cations (Maltais, Remondetto, & Subirade, 2010; Remondetto, Beyssac, & Subirade, 2004; Vazquez da Silva, et al, 2010).
Another commonly used method to form cold-set gel is altering solution pH towards protein IEP. It can be achieved by adding organic acids or acidulants, or lactic acid fermentation, which lead to the reduction of electrostatic repulsion forces between protein aggregates (Venugopal, Doke, & Nair, 2002; Riebroya, Benjakula, Visessanguanb, Eriksonc, & Rustad, 2009; Xu, Xia, Yang, Kim & Nie, 2010). Among them, glucono-δ-lactone (GDL) as an acidulant has been widely used in food products (Tseng & Xiong, 2009; Chawla, Venugopal, & Mair, 1996). GDL can be slowly hydrolyzed to gluconic acid in water, which resulted in a gradual decrease of pH to neutralize negatively charged protein aggregates and create gel with homogeneous porous structure (Malaki, Nik, Alexander, Poysa, Woodrow, & Corredig, 2011). However, the gelation mechanism and protein conformational changes at different GDL concentrations were not completely elucidated.
The gelling properties of proteins can be affected by interaction with other components, such as polysaccharides. Protein and polysaccharide are often mixed to develop food products with novel textural properties. The interactions developed among protein and polysaccharide will define the microstructure of food product and thus the resulting texture or mechanical properties. Interactions between protein and polysaccharides can be either associative or segregative depending on the molecular characteristics of the contributing polymers and the medium conditions such as pH, or ionic strength. As electrostatic interactions are produced under associative conditions between a protein and an ionic polysaccharide of opposite charge, a complex coacervate structure is obtained. When no strong interactions exist between protein and polysaccharide, interpenetrating networks are formed, where each polymer is in its own continuous network. Phase-separated networks are formed when interactions between polymers are repulsive or when there are no electrostatic forces to drive the association. This results in a bi-continuous phase or a continuous supporting phase containing inclusions of the other phase.